The long term goat of our research is the elucidation of the molecular basis of acute and chronic actions of opiates. To this end, we are studying the endogenous opioid system, with a focus on the function and regulation of opioid receptors. We hope that the results of our research will contribute knowledge toward better treatment and prevention of drug abuse. Our specific aims fall into two categories: 1) Studies of proteins that interact with opioid receptors and 2) Studies on signal transaction by opioid receptors. We have discovered several proteins that interact with the C-tail and with the third cytoplasmic loop of the mu opioid receptor. One of these is ftlamin, an actin-binding protein. We have reported that agonist-mediated mu opioid receptor regulation and trafficking are altered by the absence of filamin. We propose to identify the mechanism by which filamin acts and to determine whether the actin cytoskeleton is involved. We will extend these studies to actinin, another cytoskeletal protein we found to bind to the mu-opioid receptor Ctail. We also plan to look for proteins, which interact with the phosphorylated C-tail of the mu-receptor by changing serine, threonine and tyrosine residues, known to be phosphorylated, into aspartates, which have been found to mimic the phosphorylated state of the receptor due to their negative charge. In our studies of signaling by opioid receptors, we will explore the roles of the Rap1 and Ras pathways in MAP kinase signaling and their possible contribution to opioid tolerance and dependence. Finally, to study the effects of varying the levels of opioid receptors in transfected cells on function, trafficking and regulation, we will use a tetracycline-inducible expression system, by which the level of the mu-opioid receptors can be carefully regulated. This should provide new information and also serve to validate, or find the flaws in, the widely used model of transfected cells expressing non-physiologically high levels of opioid receptors.